Significance of mucin expression in pancreatobiliary neoplasms

Mucins are high molecular weight glycoproteins that play important roles in carcinogenesis and tumor invasion. We have described, for the first time, that pancreatic ductal adenocarcinomas (PDACs) with an aggressive behavior and a poor outcome expressed MUC1 (pan-epithelial membrane-associated mucin) but did not express MUC2 (intestinal-type secreted mucin), whereas intraductal papillary mucinous neoplasms (IPMNs) with indolent behavior and a favorable outcome did not express MUC1 but did express MUC2. These expression profiles of MUC1 and MUC2 related to the prognoses of the patients were also observed in biliary neoplasms such as intrahepatic cholangiocarcinoma (ICC)-mass-forming type (MF), mucin-producing bile duct tumor (MPBT), and extrahepatic bile duct carcinoma (EHBDC). We also found recently that high expression of MUC4 (tracheobronchial membrane-associated mucin) in PDACs, ICCs-MF, and EHBDCs was a new independent poor prognostic factor, although MUC4 was not expressed in normal pancreatobiliary tissue. High de novo expression of MUC5AC (gastric-type secreted mucin) was observed in many types of pancreatobiliary neoplasms, including all grades of pancreatic intraepithelial neoplasia (PanIN) and biliary intraepithelial neoplasia (BilIN), and all types of IPMNs and MPBTs, as well as PDACs and ICCs-MF, although MUC5AC was not expressed in normal pancreatobiliary tissue. The combined status of MUC1, MUC2, MUC4, and MUC5AC expression may be useful for the early detection of pancreatobiliary neoplasms and evaluation of their malignancy. In regard to the mechanism of mucin expression, we have recently reported that MUC1, MUC2, MUC4, and MUC5AC gene expression is regulated by epigenetics (DNA methylation and histone H3 lysine 9 modification) in cancer cell lines, including PDAC cells. Translational research of mucin gene expression mechanisms, including epigenetics, in pancreatobiliary neoplasms may give us new tools for the early and accurate detection of these neoplasms.     

Abnormal mucus in cap polyposis

Background—Cap polyposis is a rare diseasecharacterised by mucoid and bloody diarrhoea, with polyps covered by acap of mucoid and fibrinopurulent exudate. The pathogenesis is not known.
Aims—To pour some light on cap polyposispathogenesis, by examining the mucus of patients and analysing theexpression of five mucin genes, MUC2, MUC3,MUC4, MUC5AC, and MUC5B.
Patient and methods—The study was performed onbiopsy specimens taken from a patient with recurrent cap polyposis.Histochemical examination, electron microscopy, and mRNA in situhybridisation were used.
Results—The mucus of cap polyposis differed inthree respects from that of normal adult colon: abnormal ultrastructureof the mucus in the goblet cells, predominance of non-sulphated mucins, abnormal expression of the MUC4, MUC3, andMUC5AC genes.
Conclusions—Most of these abnormalities have beenreported for other pathological situations, suggesting that theabnormalities observed in the mucus of this patient with cap polyposisare probably secondary phenomena rather than primary. However, themucin abnormalities detected, which reflect deregulation of theexpression of three apomucin genes, abnormal glycosylation, andabnormalities of the secretion process, are also probably involved inthe clinical manifestations of cap polyposis.

Keywords:cap polyposis; mucins; histochemistry; ultrastructure; in situ hybridisation

     

Mucin gene expression in human embryonic and fetal intestine

Background—The intestinal epithelium is coveredby a continuous layer of mucus which is secreted by well differentiatedepithelial cells. Disregulation of the expression of mucins has beenreported to have possible implications in the neoplastic process which affects intestinal mucosae. It is well known that preneoplastic andneoplastic tissues can express fetal phenotypic characteristics.
Aims—To assess whether the expression of mucingenes in the intestinal tract is linked to the stage of cellulardifferentiation and tissue development, by studying the expression ofsix mucin genes in human fetal small intestine and colon, and alsoadult tissues.
Methods—In situ hybridisation was used to studymRNA expression of MUC2, MUC3, MUC4, MUC5B, MUC5AC, and MUC6 in 32 human embryos and fetuses (6.5-27 weeks gestation). Normal adultmucosae were used as controls.
Results—Three mucin genes, MUC2, MUC4, andMUC5AC, were differently expressed in fetal intestine compared withexpression in normal adults.
Conclusion—These differences in mucin geneexpression suggest a possible regulatory role for these products inintestinal epithelial cell differentiation.

Keywords:mucin genes; mucins; intestine; differentiation; human fetus

     

Altered Mucin Gene Expression in Stone-Containing Intrahepatic Bile Ducts and Cholangiocarcinomas

Neoplastic transformation of epithelial cells is commonly associated with alterations in the synthesis and structures of mucin. Mucin protein epitopes and mRNA levels were frequently altered in adenocarcinomas compared to corresponding normal tissues. Clinically, hepatolithiasis has been regarded as a risk factor for cholangiocarcinoma. The aims of this study were to determine the possible alteration of mucin gene expression in stone-containing intrahepatic bile ducts and cholangiocarcinomas and to try to predict whether or not hepatolithiasis has a predisposition to development of cholangiocarcinoma. In situ hybridization with DIG-tailed oligonucleotides was performed on sections of paraffin-embedded tissues of stone-containing intrahepatic bile ducts, cholangiocarcinomas, and normal controls to identify the expression of MUC2, MUC3, MUC4, MUC5B, and MUC5AC in nonneoplastic and neoplastic biliary epithelium. The findings showed that (1) while multiple diverse mucin genes were expressed in the biliary epithelium, MUC3 and MUC5B mRNA were the main mucin genes expressed in the biliary epithelium of stone-containing intrahepatic bile ducts and normal controls; (2) absent or decreased expression of MUC2, MUC3, and MUC5B of mRNA was found in cholangiocarcinomas in contrast to nonneoplastic biliary epithelium; and (3) increased expression of MUC4 and MU5AC of mRNA was found in cholangiocarcinomas and the biliary epithelium, especially for dysplastic cells of stone-containing intrahepatic bile ducts compared with normal controls. In this study, using in situ hybridization we demonstrated that neoplastic transformation of the biliary epithelium is accompained by alterations in mucin gene expression, the altered mucin gene expression in dysplastic cells of stone-containing intrahepatic bile ducts may reflect a higher potential for malignant transformation in these cells, and it could be a precursor of cholangiocarcinoma in the presence of hepatolithiasis.     

Altered Mucin Core Peptide Expression in Acute and Chronic Cholecystitis

Human mucin genes include membrane-bound mucins (MUC1, MUC3, MUC4) and secretory mucins (MUC2, MUC5AC, MUC5B, MUC6). Our aim was to determine mucin gene expression in human gallbladder cell lines, normal gallbladder from liver donors (N = 7) and surgical specimens with mild chronic cholecystitis (N = 29), chronic cholecystitis (N = 48), and acute and chronic cholecystitis (N = 27). MUC1 mRNA was ubiquitous; however, only rare MUC1 immunoreactivity was detected. MUC3, MUC5AC, MUC5B, and MUC6 mRNA were present in all gallbladder specimens and cell lines examined. Prominent MUC3, MUC5AC, MUC5B, and MUC6 immunoreactivity was present in 86–100% of normal gallbladders. The frequency of MUC5AC reactivity was decreased in specimens with acute cholecystitis (P < 0.05). In contrast, MUC2-reactivity was absent in normal gallbladder and present in 53.8% of acute cholecystitis specimens (P < 0.05). Surface epithelium is characterized by MUC3, MUC5AC, and MUC5B, whereas deeper mucosal folds display MUC5B and MUC6 immunoreactivity. Gallbladder epithelium demonstrates a unique and diverse pattern of mucin core proteins that becomes altered with increasing degrees of inflammation.     

Expression of MUC5AC, an early marker of pancreatobiliary cancer, is regulated by DNA methylation in the distal promoter region in cancer cells

Background and purpose

High de novo expression of MUC5AC (a gastric-type secreted mucin) is observed in many types of pancreatobiliary neoplasms, including precursor lesions. In this study, we show that the DNA methylation pattern is intimately correlated with MUC5AC expression in ten cancer cell lines (breast, lung, pancreas, and colon).

Methods

The CpG methylation status of the MUC5AC promoter from ?3855 to +321 was mapped using MassARRAY analysis, which utilizes base-specific cleavage of nucleic acids. ChIP assays and micro-RNA (miRNA) microarray expression profiling were also carried out in both MUC5AC-positive cells and in those with no or low MUC5AC expression.

Results

In the distal region from ?3718 to ?3670 of the promoter, MUC5AC-negative cancer cells (e.g., MDA-MB-453) were highly methylated, whereas MUC5AC-positive cells (e.g., MCF-7) had low methylation levels. The modification status of histone H3 lysine 9 (H3-K9) was also closely related to MUC5AC expression. Expression levels of miRNAs in the cancer cells were not correlated with MUC5AC expression.

Conclusion

Our results indicate that MUC5AC is regulated by CpG methylation and histone H3-K9 modification of the MUC5AC promoter distal region, but not by miRNAs. An understanding of the epigenetic regulation of MUC5AC may be of importance for the diagnosis of carcinogenic risk in the pancreatobiliary system.     

The Effect of Helicobacter pylori Eradication Treatment on the MUC 1 and Lewis Antigens Level in Human Gastric Juice: Preliminary Study

Helicobacter pylori is considered as a causative agent of gastritis, duodenal and gastric ulcers, and gastric cancer. During inflammation, association of the pathogen of gastric epithelial cells and mucins is considered important. It was postulated that Lewis b structures of secretory MUC 5AC mucin can be a receptor for the bacterium. Some authors also suggest that epithelial MUC 1 mucin may be implicated in the mechanism of infection. The main aim of our work was to support this last suggestion by evaluation of the possible changes in MUC 1 and Lewis a and b levels in gastric juice before and at the end of eradication treatment. The gastric juices of ten examined patients were chromatographed on a Sepharose 4 B column, electrotransferred on Immobilon P membranes, and assessed for MUC 1 and Lewis a and b structures using monoclonal antibodies. In 90% of examined patients, higher amounts of MUC 1 mucin were observed at the end of eradication treatment. Similar results for Lewis a and b structures were found. In the case of MUC 1 and Lewis b, the differences were statistically significant. Helicobacter pylori influences expression of the soluble form of MUC 1 mucin and Lewis a and b structures present in gastric juice.     

Mucin gene expression in intestinal epithelial cells in Crohn's disease

BACKGROUND: Crohn's disease (CD) is a chronic relapsing inflammatory bowel disease of unknown origin. It is characterised by chronic mucosal ulcerations which affect any part of the intestine but most commonly are found in the ileum and proximal colon. AIMS: Studies were undertaken to provide information regarding cell specific expression of mucin genes in the ileum of patients with CD. PATIENTS AND METHODS: Expression of mucin genes was analysed in the ileal mucosa of patients with CD and controls by in situ hybridisation and immunohistochemistry. RESULTS: In healthy ileal mucosa, patients with CD showed a pattern identical to normal controls with main expression of MUC2 and MUC3, lesser expression of MUC1 and MUC4, and no expression of MUC5AC, MUC5B, MUC6, or MUC7. In the involved mucosa, the pattern was somewhat comparable although heterogeneous to that observed in healthy ileal mucosa. Importantly, a particular mucin gene expression pattern was observed in ileal mucosa close to the ulcer margins in ulcer associated cell lineage, with the appearance of MUC5AC and MUC6 mRNAs and peptides, which are normally restricted to the stomach (MUC5AC and MUC6) and duodenum (MUC6), and disappearance of MUC2. CONCLUSIONS: Our results suggest that gel forming mucins (more particularly MUC5AC and MUC6) may have a role in epithelial wound healing after mucosal injury in inflammatory bowel diseases in addition to mucosal protection.     

Infection with Helicobacter pylori Affects All Major Secretory Cell Populations in the Human Antrum

We have investigated how gastric H. pylori infection affects antrum secretory cell types by studying the expression of secretory proteins in antrum epithelium. Antrum biopsy specimens were prospectively collected from 102 individuals (49 H. pylori-infected). Immunohistochemistry was performed for secretory mucins (MUC5AC, MUC5B, MUC6), Trefoil factor family (TFF)-peptides (TFF1, TFF2), endocrine peptides (gastrin, chromogranin A), and proliferating cells (Ki-67). Protein expression was quantified morphometrically. H. pylori infection was significantly correlated to mucosal inflammation and to epithelial atrophy and proliferation. In H. pylori-infected patients the number of proliferating cells increased significantly, and the zone of proliferating cells shifted toward the surface epithelium of the antral glands. Infection was correlated with decreased MUC5AC, TFF1, and TFF2 expression and increased MUC6 and MUC5B expression. Endocrine cells expressing chromagranin A and gastrin shifted toward the surface epithelium of the antral glands in H. pylori-infected patients. H. pylori infection and concomitant inflammation induced increased epithelial proliferation and triggered coordinate deregulation of secretory cell populations in the antrum. In particular, infection led to a coordinated increase in cells expressing MUC6 and MUC5B at the expense of MUC5AC-producing cells.     

Helicobacter pylori and gastric mucin expression: A systematic review and meta-analysis

AIM: To investigate the relationship between Helicobacter pylori (H. pylori) and mucin expression in gastric mucosa.METHODS: English Medical literature searches were conducted for gastric mucin expression in H. pylori infected people vs uninfected people. Searches were performed up to December 31^th 2014, using MEDLINE, PubMed, EMBASE, Scopus, and CENTRAL. Studies comparing mucin expression in the gastric mucosa in patients positive and negative for H. pylori infection, were included. Meta-analysis was performed by using Comprehensive meta-analysis software (Version 3, Biostat Inc., Englewood, NJ, United States). Pooled odds ratios (ORs) and 95% confidence intervals (CIs) were calculated compared mucin expression in individual studies by using the random effects model. Heterogeneity between studies was evaluated using the Cochran Q-test, and it was considered to be present if the Q-test P value was less than 0.10. I² statistic was used to measure the proportion of inconsistency in individual studies, with I² > 50% representing substantial heterogeneity. We also calculated a potential publication bias.RESULTS: Eleven studies, which represent 53 sub-studies of 15 different kinds of mucin expression, were selected according to the inclusion criteria. Every kind of mucin has been considered as one study. When a specific mucin has been studied in more than one paper, we combined the results in a nested meta-analysis of this particular mucin: MUC2, MUC6, STn, Paradoxical con A, Tn, T, Type 1 chain mucin, LeA, SLeA, LeB, AB-PAS, MUC1, and MUC5AC. The odds ratio of mucin expression in random analysis was 2.33, 95%CI: 1.230-4.411, P = 0.009, higher expression in H. pylori infected patients. Odds ratio for mucin expression in H. pylori positive patients was higher for MUC6 (9.244, 95%CI: 1.567-54.515, P = 0.014), and significantly lower for MUC5AC (0.447, 95%CI: 0.211-0.949, P = 0.036). Thus, H. pylori infection may increase MUC6 expression and decrease MUC5AC expression by 924% and 52%, respectively.CONCLUSION: H. pylori inhibits MUC5AC expression in the gastric epithelium, and facilitates colonization. In contrast, increased MUC6 expression may help inhibiting colonization, using MUC6 antibiotics properties.     

Distinctive expression of CD133 between intraductal papillary neoplasms of the bile duct and bile duct adenocarcinomas

Aim: Intraductal papillary neoplasm of the bile duct (IPNB), a novel entity of biliary disease, is recently advocated as the counterpart of pancreatic intraductal papillary mucinous neoplasm (IPMN) because both are in common with a large amount of mucin production and papillary growth. Based on our recent finding that expression of CD133, a cancer stem cell marker, is lacking in pancreatic IPMN, we herein focused on CD133 expression of IPNB in comparison with intrahepatic cholangiocellular carcinoma (IHCCC) or hilar bile duct cancer (HBDC). Methods: Expression of CD133 protein was immunohistochemically determined in patients with IPNB (n = 7), IHCCC (n = 16) or HBDC (n = 8). In addition, morphological and immunohistochemical mucin expression patterns were characterized in IPNB, and clinicopathological features including prognosis were compared between IPNB and other biliary tumors. Results: The IPNB group included significantly more females than the other two groups, and had a longer survival time. While no CD133 expression was observed in IPNB tumor, 16.4% of cancer cells in IHCCC and 17.2% of cells in HBDC expressed CD133. Among seven patients with IPNB, six (86%) were morphologically the pancreatobiliary type and four of six showed mucin expression pattern of the typical pancreatobiliary type (MUC1+/MUC2‐/MUC5AC+). Conclusion: Loss of CD133 expression supports the hypothesis that IPNB is a counterpart of pancreatic IPMN with a differing carcinogenesis from conventional bile duct adenocarcinomas.     

Intraductal neoplasm of the intrahepatic bile duct: clinicopathological study of 24 cases

AIM: To investigate the clinicopathological features of intraductal neoplasm of the intrahepatic bile duct (INihB). METHODS: Clinicopathological features of 24 cases of INihB, which were previously diagnosed as biliary papillomatosis or intraductal growth of intrahepatic biliary neoplasm, were reviewed. Mucin immunohistochemistry was performed for mucin (MUC)1, MUC2, MUC5AC and MUC6. Ki-67, P53 and β-catenin immunoreactivity were also examined. We categorized each tumor as adenoma (low grade), borderline (intermediate grade), and malignant (carcinoma in situ , high grade including tumors with microinvasion). RESULTS: Among 24 cases of INihB, we identified 24 tumors. Twenty of 24 tumors (83%) were composed of a papillary structure; the same feature observed in intraductal papillary neoplasm of the bile duct (IPNB). In contrast, the remaining four tumors (17%) showed both tubular and papillary structures. In three of the four tumors (75%), macroscopic mucin secretion was limited but microscopic intracellular mucin was evident. Histologically, 16 tumors (67%) were malignant, three (12%) were borderline, and five (21%) were adenoma. Microinvasion was found in four cases (17%). Immunohistochemical analysis revealed that MUC1 was not expressed in the borderline/adenoma group but was expressed only in malignant lesions (P = 0.0095). Ki-67 labeling index (LI) was significantly higher in the malignant group than in the borderline/adenoma group (22.2 ± 15.5 vs 7.5 ± 6.3, P 0.01). In the 16 malignant cases, expression of MUC5AC showed borderline significant association with high Ki-67 LI (P = 0.0622). Nuclear expression of β-catenin was observed in two (8%) of the 24 tumors, and these two tumors also showed MUC1 expression. P53 was negative in all tumors. CONCLUSION: Some cases of INihB have a tubular structure, and are subcategorized as IPNB with tubular structure. MUC1 expression in INihB correlates positively with degree of malignancy.     

Intestinal Goblet Cells and Mucins in Health and Disease: Recent Insights and Progress

The mucus layer coating the gastrointestinal tract is the front line of innate host defense, largely because of the secretory products of intestinal goblet cells. Goblet cells synthesize secretory mucin glycoproteins (MUC2) and bioactive molecules such as epithelial membrane-bound mucins (MUC1, MUC3, MUC17), trefoil factor peptides (TFF), resistin-like molecule β (RELMβ), and Fc-γ binding protein (Fcgbp). The MUC2 mucin protein forms trimers by disulfide bonding in cysteine-rich amino terminal von Willebrand factor (vWF) domains, coupled with crosslinking provided by TFF and Fcgbp proteins with MUC2 vWF domains, resulting in a highly viscous extracellular layer. Colonization by commensal intestinal microbiota is limited to an outer “loose” mucus layer, and interacts with the diverse oligosaccharides of mucin glycoproteins, whereas an “inner” adherent mucus layer is largely devoid of bacteria. Defective mucus layers resulting from lack of MUC2 mucin, mutated Muc2 mucin vWF domains, or from deletion of core mucin glycosyltransferase enzymes in mice result in increased bacterial adhesion to the surface epithelium, increased intestinal permeability, and enhanced susceptibility to colitis caused by dextran sodium sulfate. Changes in mucin gene expression and mucin glycan structures occur in cancers of the intestine, contributing to diverse biologic properties involved in the development and progression of cancer. Further research is needed on identification and functional significance of various components of mucus layers and the complex interactions among mucus layers, microbiota, epithelial cells, and the underlying innate and adaptive immunity. Further elucidation of the regulatory mechanisms involved in mucin changes in cancer and inflammation may lead to the development of novel therapeutic approaches.